18 different population of cells in different developmental stages in hematopoietic hierarchy have been purifyed by FACS analyses from wild type C57Bl6 mice and subjected to Micrroarray Affymetrix mouse 430.2 platform
CCAAT/enhancer binding protein alpha (C/EBP(alpha))-induced transdifferentiation of pre-B cells into macrophages involves no overt retrodifferentiation.
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View SamplesEarlier work has shown that pre-B cells can be converted into macrophages by the transcription factor C/EBP? at very high frequencies. Using this system we have now performed a systematic analysis of the question whether during transdifferentiation the cells transiently reactivate progenitor restricted genes or even retrodifferentiate. A transcriptome analysis of transdifferentiating cells showed that most genes are continuously up or downregulated, acquiring a macrophage phenotype within 5 days. In addition, we observed the transient reactivation of a subset of immature myeloid markers, as well as low levels of the progenitor markers Kit and Flt3 and a few lineage inappropriate genes. However, we were unable to observe the re-expression of cell surface marker combinations that characterize hematopoietic stem and progenitor cells (HSPCs), including c-Kit and Flt3. This was the case even when C/EBPalpha was activated in pre-B cells under culture conditions that favor HSPC growth or when the transcription factor was activated in a time limited fashion. Together, our findings are consistent with the notion that the conversion from pre-B cells to macrophages is mostly direct and does not involve overt retrodifferentiation.
CCAAT/enhancer binding protein alpha (C/EBP(alpha))-induced transdifferentiation of pre-B cells into macrophages involves no overt retrodifferentiation.
Specimen part, Time
View SamplesThe major myeloid blood cell lineages, including erythrocytes, platelets, granulocytes and macrophages, are generated from hematopoietic stem cells (HSC) by differentiation through a series of increasingly more committed progenitor cells. Precise phenotypic identification and functional characterization of such intermediate progenitors has important consequences for understanding fundamental differentiation processes and is clinically relevant since such events become dysregulated in various disease settings, including leukemia. While previous studies have suggested a hierarchy for myeloid differentiation involving a common progenitor through which all myeloid lineages are derived, several recent studies have suggested that such a developmental intermediate might not be an absolute requirement. Here, we evaluated the functional in vitro and in vivo potentials of a range of prospectively isolated myeloid precursors with differential expression of CD150, Endoglin and CD41. Our studies reveal a complex hierarchy of myeloerythroid progenitors with distinct and developmentally restricted lineage potentials. Global gene expression signatures of these cellular subsets revealed expression patterns consistent with their functional capacities, while hierarchical clustering analysis provides details on their lineage relationships. These data challenge existing models of hematopoietic differentiation, by suggesting that progenitors of the innate and adaptive immune system in the adult separate late, and to a large extent, following the divergence of megakaryocytic/erythroid potential.
Elucidation of the phenotypic, functional, and molecular topography of a myeloerythroid progenitor cell hierarchy.
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View SamplesFull title: Genomics based analysis of interactions between developing B-lymphocytes and stromal cells reveal complex interactions and two-way communication
Genomics based analysis of interactions between developing B-lymphocytes and stromal cells reveal complex interactions and two-way communication.
Specimen part
View SamplesNIH-3T3 cells transduced with either EBF1-, PPARg2- or empty vector were stimulated with hormones to initiate adipocyte differentiation. RNA extraction was done using TriZol at d0, d2, d4 and d10 after stimulation. Samples were handled according to standard affymetrix protocols.
Gene expression analysis suggests that EBF-1 and PPARgamma2 induce adipogenesis of NIH-3T3 cells with similar efficiency and kinetics.
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View SamplesEbf1 is a transcription factor with documented, and dose dependent, functions in both normal and malignant B-lymphocyte development. In order to understand more about the role of Ebf1 in malignant transformation, we have investigated the impact of reduced functional Ebf1 dose on early B-cell progenitors. Gene expression analysis in loss and gain of function analysis suggested that Ebf1 was involved in the regulation of genes of importance for DNA repair as well as cell survival. Investigation of the level of DNA damage in steady state as well as after induction of DNA damage by UV light supported that pro-B cells lacking one functional allele of Ebf1 display a reduced ability to repair DNA damage. This was correlated to a reduction in expression of Rad51 and combined analysis of published 4C and chromatin Immuno precipitation data suggested that this gene is a direct target for Ebf1. Even though the lack of one allele of Ebf1 did not result in any dramatic increase of tumor formation, we noted a dramatic increase in the formation of pro-B cell leukemia in mice carrying a combined heterozygote mutation in the Ebf1 and Pax5 genes. Even though the tumors were phenotypically similar and stable, we noted a large degree of molecular heterogeneity well in line with a mechanism involving impaired DNA repair. Our data support the idea that Ebf1 controls homologous DNA repair in a dose dependent manner and that this may explain the frequent involvement of Ebf1 in human leukemia
Ebf1 heterozygosity results in increased DNA damage in pro-B cells and their synergistic transformation by Pax5 haploinsufficiency.
Specimen part, Cell line, Time
View SamplesLineage negativ Sca1+ Kit+ bone marrow cells (containing putative hematopoietic stem cells) subfractionation based on CD34 and FLT3 identifies three functionally destinc subpopulations (LSKCD34-FLT3-, LSKCD34+FLT3- & LSKCD34+FLT3+).
Molecular evidence for hierarchical transcriptional lineage priming in fetal and adult stem cells and multipotent progenitors.
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View SamplesThe instrinsic regenerative capacity of human fetal cardiac mesenchymal stromal cells (MSCs) has not been fully characterised. Here we demonstrate that we can expand cells with characteristics of cardiovascular progenitor cells from the MSC population of human fetal hearts with only minor fluctuations over time in culture (from day 15 to day 48).
Wnt/β-Catenin Stimulation and Laminins Support Cardiovascular Cell Progenitor Expansion from Human Fetal Cardiac Mesenchymal Stromal Cells.
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